Point-of-Care and Dual-Response Detection of Hydrazine/Hypochlorite-Based on a Smart Hydrogel Sensor and Applications in Information Security and Bioimaging

A novel dual-response fluorescence probe (XBT-CN) was developed by using a fluorescence priming strategy for quantitative monitoring and visualization of hydrazine (N2H4) and hypochlorite (ClO−). With the addition of N2H4/ClO−, the cleavage reaction of C=C bond initiated by N2H4/ClO− was transformed into corresponding hydrazone and aldehyde derivatives, inducing the probe XBT-CN appeared a fluorescence “off-on” response, which was verified by DFT calculation. HRMS spectra were also conducted to confirm the sensitive mechanism of XBT-CN to N2H4 and ClO−. The probe XBT-CN had an obvious fluorescence response to N2H4 and ClO−, which caused a significant color change in unprotected eyes. In addition, the detection limits of XBT-CN for N2H4 and ClO− were 27 nM and 34 nM, respectively. Interference tests showed that other competitive analytes could hardly interfere with the detection of N2H4 and ClO− in a complex environment. In order to realize the point-of-care detection of N2H4 and ClO−, an XBT-CN@hydrogel test kit combined with a portable smartphone was developed. Furthermore, the portable test kit has been applied to the detection of N2H4 and ClO− in a real-world environment and food samples, and a series of good results have been achieved. Attractively, we demonstrated that XBT-CN@hydrogel was successfully applied as an encryption ink in the field of information security. Finally, the probe can also be used to monitor and distinguish N2H4 and ClO− in living cells, exhibiting excellent biocompatibility and low cytotoxicity.

the field of information security. Finally, XBT-CN has been practically applied to biological imaging of N 2 H 4 and ClO − in living cells (Figure 1).

Design and Synthesis of the Probe XBT-CN
XBT-CN probe was designed with xanthone as a fluorescent group and benzothiazole acetonitrile as a recognition unit. Xanthone is chosen in this paper because of its high emission efficiency and simple preparation process. Benzothiazole acetonitrile, as a typical recognition group of N 2 H 4 , is a good reaction site for recognizing N 2 H 4 due to C=C bridging group and is introduced into xanthone derivative to detect N 2 H 4 via a nucleophilic reaction to form a yellow hydrazone derivative. In addition, the strong electron withdrawing group of benzothiazole acetonitrile makes the C=C bond easier to be attacked by ClO − , which leads to the cancellation of the conjugated system and the formation of aldehyde conjugated product of the oxidative nucleophilic reaction. The design strategy of XBT-CN reacts with N 2 H 4 and ClO − to produce two different products, which may lead to a fluorescence "on-off" strategy with different fluorescence signals. In Scheme 1, the synthesis process of probe XBT-CN is given. Firstly, at room temperature, cyclohexanone reacts with PBr 3 in CH 2 Cl 2 to give compound 1. Compound 2 was obtained by refluxing 2-hydroxy-4-methoxybenzaldehyde with compound 1 in DMF. Then, there was a condensation reaction occurred between compound 2 and 2-benzothiazole acetonitrile (3) in the presence of the catalytic amount of piperidine, and the probe XBT-CN was obtained as a dark blue solid. The probe XBT-CN and related intermediates were confirmed by 1 H NMR, 13 C NMR, and HRMS (Figures S1-S12).

UV Response of XBT-CN to N 2 H 4 and ClO −
The sensing properties of probe XBT-CN for N 2 H 4 and ClO − in CH 3 OH/PBS solution (v/v = 8:2, pH = 7.4, 10 mM) were determined for the first time by UV-Vis and fluorescence spectra. As can be seen from Figure 2a, the UV-Vis spectra of XBT-CN (10 µM) have two absorption peaks at 312 nm and 523 nm, respectively. With the addition of N 2 H 4 (0-100 µM), the maximum absorption peaks at 312 nm and 523 nm decreased, and the peak at 384 nm increased gradually. Simultaneously, the modena color of the XBT-CN solution gradually changes to yellow under sunlight, which can be observed by the naked eye (Figure 2a, inset). As shown in Figure 2c, we also investigated the UV-Vis absorption of ClO − by XBT-CN. After ClO − (0-100 µM) was added, the absorption peaks of XBT-CN at 312 nm and 523 nm gradually decreased with the peak at 389 nm increased, and the color of the XBT-CN solution changed from a modena color to colorless under sunlight, which was beneficial to naked-eye monitoring of ClO − (Figure 2c, inset). Furthermore, we also studied the color changes of XBT-CN solution with the addition of different concentrations of N 2 H 4 and ClO − under sunlight (Figure 2e,f), and these results significantly show that the probe XBT-CN can provide visual detection of N 2 H 4 and ClO − by naked eyes.

Fluorescence Response of XBT-CN to N 2 H 4 and ClO −
The fluorescence response of XBT-CN (10 µM) in mixed solution (CH 3 OH/PBS, v/v = 8/2, pH = 7.4) with or without N 2 H 4 /ClO − was also investigated. As shown in Figure 3a, XBT-CN had a weak fluorescence intensity at 470 nm. After adding different concentrations of N 2 H 4 (0-100 µM), the fluorescence intensity at 470 nm increased gradually until it reached the maximum at 100 µM. This change made the fluorescence of XBT-CN change from quenched to luminous yellow, which was witnessed by the naked eye under a 365 nm lamp (Figure 3e). In addition, in the range of N 2 H 4 concentration from 0 to 100 µM, the emission intensity has a good linear relationship with N 2 H 4 concentration (R 2 = 0.9978), and the LOD is 27 nM (Figure 3b). In addition, the responses of XBT-CN and ClO − were also studied. As exhibited in Figure 3c, when different concentrations of ClO − (0-100 µM) were added, the fluorescence intensity of XBT-CN increased at 490 nm, which inducing the fluorescence of XBT-CN changed from quenched to brilliant blue. This change was also observed under a 365 nm lamp (Figure 3f). In addition, in the range of 0-100 µM, the relationship curve between fluorescence intensity and ClO − concentration (R 2 = 0.9977) shows a good linear relationship, and the LOD is as low as 34 nM (Figure 3d). These results indicate that XBT-CN has high sensitivity in fluorescent detection of N 2 H 4 and ClO − , providing a practical strategy for environmental safety assessment and biological sample detection.

Selectivity and Anti-Interference Performance of XBT-CN
In order to evaluate the selectivity of XBT-CN to N 2 H 4 and ClO − , the fluorescence performance of the probe in the presence of all kinds of interference was recorded. As shown in Figure 4a, the selectivity of probe XBT-CN to N 2 H 4 was judged by using various analytes, such as anions, metal ions and amine. Except for N 2 H 4 , other analytes cannot cause obvious enhancement of fluorescence intensity. In addition, in the system where N 2 H 4 coexists with other analytes (Figure 4b), the fluorescence response of XBT-CN to N 2 H 4 was almost undisturbed. On the contrary, N 2 H 4 significantly increased the fluorescence intensity of XBT-CN in CH 3 OH/PBS solution (70 times). We also obtained similar results, that is, XBT-CN showed high selectivity to ClO − . As described in Figure 4c,d, various analytes, such as anions, metal ions and other oxidants, did not lead to apparent enhancement of fluorescence intensity except for ClO − . These results demonstrate that XBT-CN has good specificity for the detection of N 2 H 4 and ClO − in actual environment and complex physical environment.

Effect of pH and Response Time
In the practical application of probes, pH value is an important influencing factor. The effect of pH on the fluorescence response of XBT-CN in the presence and absence of N 2 H 4 and ClO − was studied. As shown in Figure S13, in the range of pH 1.0-14.0, the fluorescence intensity of the probe itself has little change. This result suggests that the properties of XBT-CN are stable in a wide pH range and can be applied to detect the complex samples. Afterward, we recorded the fluorescence changes of XBT-CN treated with N 2 H 4 or ClO − . When the probe was incubated with N 2 H 4 or ClO − , the fluorescence intensity changed obviously in the range of pH 1.0-7.0, and reached the maximum in the range of pH 7.0-8.0. The fluorescence intensity under strong acidity in the pH range of 1 to 5 is weak, which may be the reason for the protonation of the analyte. Another reason may due to hydrazine's strong alkalinity mainly existing in the form of [N 2 H 5 ] + under acidic conditions and loss of nucleophilicity. However, with the increase in pH value in the range of 9 to 14, the fluorescence intensity decreased obviously. This phenomenon may due to the nucleophilic attack of OH − on XBT-CN under strong alkaline conditions, which disturb the nucleophilic attack on XBT-CN by N 2 H 4 or ClO − . The results show that the probe XBT-CN has a good response to N 2 H 4 and ClO − in the range of pH 7.0-8.0, and can be hopeful for the detection of N 2 H 4 and ClO − in biological system.  With the above promising results in hand, the time-dependent fluorescence responses of XBT-CN toward N 2 H 4 and ClO − were further studied in CH 3 OH/PBS (v/v = 8:2, pH = 7.4) at room temperature. As revealed in Figure S14, there were almost no fluorescence intensity at both 470 nm and 490 nm in the solution when without N 2 H 4 or ClO − . However, with the addition of N 2 H 4 , the fluorescence intensity of XBT-CN at 470 nm promptly enhanced and reached a plateau at 20 s. Meanwhile, the fluorescence intensity of XBT-CN at 490 nm increased gradually in the presence of ClO − and became saturated at 15 s. These results strongly suggest that the rapid response probe XBT-CN can realized the real-time detection of N 2 H 4 and ClO − . Further kinetic studies also showed that the calculated pseudo-first-order rate constant (k') for detecting of N 2 H 4 and ClO − was 0.3656 min −1 and 0.3374 min −1 , respectively (Figures S16 and S17). All the above results demonstrated that XBT-CN is a promising fluorescent probe, and could detect N 2 H 4 and ClO − in quantitative and an expressed speed by the fluorescence spectrometry method.

Sensing Mechanism Study of XBT-CN to N 2 H 4 and ClO −
For purpose of determine the reaction mechanism, we investigated the reaction of XBT-CN with N 2 H 4 and ClO − on the basis of HRMS analysis. HRMS spectra of XBT-CN in CH 3 OH, N 2 H 4 , and ClO − for 30 min were recorded respectively. As shown in Figure S15A, XBT-CN (ε = 3.39 × 10 3 M −1 cm −1 , Φ = 0.002) itself showed a peak at m/z 398.1163. When XBT-CN (10 µM) is treated with N 2 H 4 (100 µM), the original peak at m/z 398.1163 disappeared and a significant peak is observed at m/z 256.1286, which is consistent with the value estimated with response product XBT-CN-N 2 H 4 (expected [M + H] + at 256.1212, Figure S16B). This spectral change shows that a hydrazone derivative XBT-CN-N 2 H 4 is formed through the nucleophilic addition reaction between the probe and N 2 H 4 , and the reaction mechanism is illustrated in Scheme 2. Furthermore, we also analyzed the reaction product XBT-CN-ClO − -adduct ( Figure S16C). The product was separated and studied by HRMS. The peak at 242.1017 m/z is consistent with the expected formylation product (expected [M + H] + 242.0943). The reasonable mechanism between XBT-CN and ClO − is also described in Scheme 1. The C=C bond of XBT-CN was first attacked by ClO − , and then cleaved by ClO − oxidation to form an aldehyde derivative (XBT-CN-ClO − ). Thus it can be seen, these results prove that the products produced by the reaction of probe XBT-CN with N 2 H 4 and ClO − are expected XBT-CN-N 2 H 4 (ε = 7.12 × 10 4 M −1 cm −1 , Φ = 0.46) and XBT-CN-ClO − (ε = 9.74 × 10 4 M −1 cm −1 , Φ = 0.39), respectively. The proposed sensing process was further inspected via HPLC analysis. As is shown in Figure 5a, the probe XBT-CN displayed a main peak with retention times (t R ) of 14.47 min. After the incubation of XBT-CN (10 µM) with N 2 H 4 (10 µM), a new peak at 4.51 min appeared, which was corresponded to that of XBT-CN-N 2 H 4 . Incubating XBT-CN (10 µM) with high concentration of N 2 H 4 (100 µM) leads to a chromatographic peak identical to that of synthetic XBT-CN-N 2 H 4 standard, indicating the complete conversion of XBT-CN to XBT-CN-N 2 H 4 . In addition, when XBT-CN (10 µM) reacted with ClO − (10 µM), a new peak at 4.51 min appeared, which was consistent with the peak of XBT-CN-ClO − (Figure 5b). Furhtermore, XBT-CN (10 µM) reacting with high concentration of ClO − (100 µM) leads to a chromatographic peak corresponding to that of synthetic XBT-CN-ClO − standard, indicating the complete conversion of XBT-CN to XBT-CN-ClO − . These HPLC results evidently confirmed that the sensing mechanism which is consistent with the HRMS results. The interaction mechanism of XBT-CN with N 2 H 4 and ClO − was further confirmed by DFT calculations. The molecule structures of XBT-CN and the response products to N 2 H 4 and ClO − were calculated by density functional theory at B3LYP/6-31 G(d) level using Gaussian 09 program. As shown in Figure 6, when reacted with N 2 H 4 , the optimized configurations of XBT-CN and XBT-CN-N 2 H 4 are acquired. Their energies of HOMO and LUMO orbits are also calculated. Furthermore, the energy gap of HOMO-LUMO orbit of XBT-CN was 1.5728 eV. After reacting with N 2 H 4 and ClO − , the energy gaps of HOMO-LUMO orbit of XBT-CN-N 2 H 4 and XBT-CN-ClO − were 1.9241 eV and 2.1852 eV respectively, both higher than that of XBT-CN. This result may be because of the generation of C=N and C=O bond from the nucleophilic and oxidation reaction of C=C induced by N 2 H 4 and ClO − , respectively. The above calculation results are completely in agreement with the spectral analysis, further demonstrating the proposed sensing mechanism of the probe XBT-CN.

Point-of-Care Detection of N 2 H 4/ ClO − by Hydrogel Test Kit with Smartphone
To achieve the point-of-care detection of N 2 H 4 and ClO − , a portable XBT-CN@hydrogel test kit was fabricated by placing agarose loaded with XBT-CN in a lid of centrifuge tube. The detection procedure for N 2 H 4 and ClO − by the portable test kit was displayed in Figure 7a. Moreover, the smartphone can photograph pictures of the fluorescence images and then exhibit values of R (red), G (green), and B (blue) to respond to different samples promptly. As could be seen from Figure 7b, with the addition of different concentrations of N 2 H 4 or ClO − , the fluorescence colors of the hydrogel test kit displayed a range of changes under UV light. After that, the color-analysis smartphone app analyzed the colors and immediately output the corresponding values of RGB. Interestingly, the RGB values was interconnected with the concentration of N 2 H 4 or ClO − . In the N 2 H 4 titration experiment, there was a good linear relationship between (R+G)/B value and N 2 H 4 concentration (R 2 = 0.9961) in the range of 0-40 µM, and the LOD was calculated to be 19 nM (Figure 7c). Similarly, in the titration experiment of ClO − , there was also a good linear relationship existed between (R+G+B)/B value and ClO − concentration (R 2 = 0.9878) within the range of 0-30 µM, and the LOD was calculated to be 28 nM. The LODs are lower than previous fluorescence methods based on bulky instruments, revealing the excellent detection sensitivity of this portable test kit. Thus it can be seen, the XBT-CN@hydrogel test kit combined smartphone holds a great potential to be a portable tool for point-of-care detection of N 2 H 4 and ClO − .

Application in Real Samples
Inspired by the above results, N 2 H 4 and ClO − in actual samples were detected by the XBT-CN@hydrogel test kit to demonstrate their practicability. We mainly focus on real samples in the environment (such as water and soil) and food samples. Environmental samples include soil (from cropland, wetlands, and sandland) and water (from tap water, lake water, and river water). Food samples include rice, flour, vegetables, and drinks. All the above species are might under the pressure of N 2 H 4 or ClO − risks. Firstly, the test solution of the actual sample was prepared, and then exogenous N 2 H 4 and ClO − with different concentrations (5.0 µM and 10.0 µM) were added. Then the sample solution was dropped into the hydrogel that was placed on the lid of the centrifuge tube and placed upside down for 15 min. Finally, the fluorescent color photos of the lids were photographed and input into the smartphone. According to the above linear regression equation, the levels of N 2 H 4 and ClO − in different samples were obtained ( Figure 8). Obviously, the XBT-CN@hydrogel test kit could quantitatively detect N 2 H 4 and ClO − in real samples. In order to further evaluate the practical application ability of the XBT-CN@hydrogel test kit in various practical samples, different concentrations of N 2 H 4 and ClO − in water, soil, and food were further determined by the probe XBT-CN in two methods. As shown in Tables S2-S7, the detection results are consistent with the actual addition of N 2 H 4 or ClO − in each sample by either the spectroscopic method or hydrogel test kit. Furthermore, the results showed that the recoveries ranged from 97% to 106%. The experimental results prove that XBT-CN can be used for the determination of N 2 H 4 and ClO − in various real samples and that the spectroscopic method and the hydrogel test kit have high accuracy and reliability for the quantitative determination of N 2 H 4 and ClO − in the environment and food.

Analysis of ClO − in MPO/H 2 O 2 /Cl − System
In order to evaluate the ability to detect ClO − generated enzymatically employing MPO, the control experiment was conducted, as shown in Figure S18. After the addition of XBT-CN to the MPO/H 2 O 2 /Cl − system, a rapid fluorescence intensity response was observed. The fluorescence growth for XBT-CN lasted for 5 min and then became steady. As depicted in Figure S18, the formation of product XBT-CN-ClO − occurred only when MPO, NaCl, and H 2 O 2 were present in the reaction mixture. The addition of catalase, which is responsible for the decomposition of hydrogen peroxide to water and oxygen, prevented the formation of ClO − , and thus oxidation of XBT-CN to XBT-CN-ClO − . The absence of any component necessary for the enzymatic production of HOCl resulted in the lack of formation of XBT-CN-ClO − . It was also proved that the XBT-CN probe is able to detect MPO activity in real time.

Role as Encryption Ink in Data Security
Encouraged by the strong fluorescence visual response of XBT-CN@hydrogel to N 2 H 4 /ClO − accompanied by its excellent stability, liquidity, and injectability, we found that XBT-CN@hydrogel can be used as encryption ink in the field of information security. As shown in Figure 9, the number "0611" was written on a thin glass slide with XBT-CN@hydrogel. In the beginning, the numbers were invisible with weak fluorescence under UV light. However, the numbers can be illuminated immediately and send out luminous yellow fluorescence visualization in 3 s by spraying a N 2 H 4 solution. Similarly, once the NaClO solution was applied, the number "0611" displayed a strong blue fluorescence. In the absence and presence of N 2 H 4 /ClO − , the XBT-CN@hydrogel always presented achromatous under sunlight. Therefore, XBT-CN@hydrogel has a broad prospect to be used as an encryption ink in the field of data security.

Fluorescence Imaging in Living Cells
The above experiments initially proved that XBT-CN could be used to detect N 2 H 4 and ClO − in a complex system, and the detection ability of the XBT-CN probe in living cells was further investigated. Firstly, the toxicity of XBT-CN to GL261 cells (mouse glioma cells) was evaluated by standard MTT assay. After being treated with different concentrations of XBT-CN for 24 h, the cell survival rate was over 94%, as shown in Figure S19, which indicated that XBT-CN had low cytotoxicity and excellent biocompatibility in living cells. Afterward, the imaging ability of the probe to N 2 H 4 and ClO − in living cells was evaluated by fluorescence microscope. GL261 cells were incubated with XBT-CN (20 µM) for 2 h, and intracellular red fluorescence was observed (Figure 10c). XBT-CN treated cells were then incubated with N 2 H 4 (80 µM) or ClO − (100 µM) for 2 h. As exhibited in Figure 10e, the red fluorescence at the red channel was weakened, and the green fluorescence at the green channel was obvious, which indicated that the C=C double bond of XBT-CN was transformed into hydrazone by N 2 H 4 , which caused the green fluorescence to increase. On the other hand, we also obtained a clear blue fluorescence signal in cells under a fluorescence microscope, indicating that ClO − can completely oxidize the C=C double bond of XBT-CN into aldehyde and induce the fluorescence of living cells to change from red to blue (Figure 10g). These results indicate that XBT-CN possesses low cytotoxicity and excellent biocompatibility and further can be used as a reliable and powerful tool for the detection and visualization of N 2 H 4 and ClO − in living cells.

Materials and Instruments
All chemicals and solvents were purchased from commercial sources and used without further purification. Solvents were purified by standard procedures. The water was purified by a Millipore filtration system. All chemical reactions were detected by thinlayer chromatography under 254 nm (or 365 nm) UV lamp. 1 H and 13 C NMR spectra were recorded on a WIPM 400 spectrometer using tetramethylsilane (TMS) as the internal standard. High-resolution mass (HRMS) spectra were recorded on a Waters LCT Premier XE spectrometer using standard conditions (ESI, 70 eV). All absorption spectrums were measured on a UV-1800 UV visible spectrophotometer. All fluorescence spectra were measured on an FL-4500 fluorescence spectrometer. The pH measurements were taken on a METTLER TOLEDO FiveEasy Plus pH meter. The cell imaging experiments were taken under an Olympus IX71 inverted fluorescence microscope.

Synthesis
As illustrated in Scheme 1, the probe XBT-CN was synthesized through a process including four steps. The structures of all the related intermediates and probe were confirmed by 1 H NMR, 13 C NMR, and HRMS spectra (Figure S1-S12).

Synthesis of Compound 1
The mixture of DMF (4.5 mL, 60.0 mmol) and CH 2 Cl 2 (30 mL) was kept at 0 • C. Then PBr 3 (3.8 mL, 60.0 mmol) was slowly added into the mixture and stirred for 1.0 h at 0 • C. After that, cyclohexanone (1.7 mL, 20.0 mmol) was dropwise added into the mixture and stirred for 16 h at room temperature. After the reaction was completed, the mixture was slowly poured into ice water (100 mL), and NaHCO 3 powder was slowly added to the mixture until the pH = 7. The solution was extracted with CH 2 Cl 2 , and the organic layer was dried with anhydrous Na 2 SO 4 for 8 h. Finally, the solvent was evaporated to give 1 as a yellow oil (3.20 g, 85 % yield). 1

Synthesis of Compound 2
Compound 1 (0.47 g, 2.5 mmol) and 2-hydroxy-4-methoxybenzaldehyde (0.32 g, 2.0 mmol) were dissolved in DMF (10 mL), and Cs 2 CO 3 (1.89 g, 6.0 mmol) were added to the mixture subsequently. The reaction mixture was stirred for 12 h at room temperature and then filtered and concentrated. The concentration was dissolved in CH 2 Cl 2 (50 mL) and was washed with pure water (30 mL × 3). The organic layer was dried with anhydrous Na 2 SO 4 for 8 h and then filtered. Afterward, the solvent was concentrated, and the obtained crude product was further purified by silica gel column chromatography (PE/EA, v/v = 15:1) to afford 2 as a yellow solid with a yield of 77% (0.37 g). 1 13

Synthesis of Compound 3
The mixture of o-aminophenol (0.18 g, 1.0 mmol) and malononitrile (0.26 g, 1.2 mmol) in EtOH (10 mL) was refluxed for 9 h. Furthermore, the reaction solution was concentrated after the reaction was completed. Then the concentration was further purified by column chromatography (PE/EA, v/v = 20:1) to afford 3 as a white solid (0.15 g, 88 % yield). 1

Synthesis of Compound XBT-CN
Firstly, compound 2 (0.18 g, 1.0 mmol) and compound 3 (0.24 g, 1.2 mmol) were dissolved in 10 mL EtOH. Later, piperidine (0.1 mL) was dropped into the mixture, and the solution was refluxed for 12 h. Furthermore, the reaction solution was concentrated after the reaction was completed. Then the concentration was further purified by column chromatography (PE/EA, v/v = 30:1) to afford XBT-CN as a blue solid (0.21 g, 53 % yield). 1

Spectroscopic Measurements
Stock solution of the probe XBT-CN (1 mM) were prepared in CH 3 OH. Stock solutions of N 2 H 4 , ClO − , and other interfering analytes (10 mM) were all prepared in ultrapure water. All the fluorescence measurements were performed by scanning the spectra in the range of 200 nm and 700 nm and excited at the wavelength of 440 nm. The slit widths for both excitation and emission spectra were 5 nm. All fluorescence spectra were excited at 440 nm and acquired in CH 3 OH/PBS solution (v/v = 8:2, pH = 7.4, 10 mM) at room temperature.

Determination of the Fluorescence Quantum Yield
The fluorescence quantum yields of XBT-CN, XBT-CN-N 2 H 4 , and XBT-CN-ClO − were determined with quinine sulfate (Φ = 0.54 in 0.1 M H 2 SO 4 ) as a fluorescence standard. The quantum yields were calculated using the following equation: Φ r = Φ s (F r A s /F s A r )(n r /n s ) 2 Φ r and Φ s denote the quantum yield of the samples and standard sample, respectively. F, A, and n denote the region of the emission band, absorbance, and refractive index of the solvent, respectively. Subscripts s and r refer to the standard and the unknown, respectively.

HPLC Analysis
HPLC analysis was carried out on a Waters 2695 Alliance system (Milford, MA, USA) equipped with a quaternary solvent delivery system, a column oven, an auto-sampler, and a photodiode array detector. The analytes were separated with a C18 column (Welchrom, 150 mm × 4.6 mm, 5.0 µm). The flow rate was 1.0 mL/min. The eluent components were water (A) and acetonitrile (B). The mobile phase gradient was as follows: the proportion of Phase B increased from 10 to 80% over 10 min at the flow rate of 1.0 mL/min. The detection wavelength was set at 365 nm.

Density Functional Theory
According to the basis set of B3LYP/6-31 G(d) [46][47][48][49], after responding to N 2 H 4 and ClO − , the geometrical configuration of the probe XBT-CN in the ground state and excited state were respectively calculated and optimized by density functional theory (DFT). Moreover, DFT can be applied to calculate the electron cloud distribution and energy gaps of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) state.

Preparation of the XBT-CN@Hydrogel Portable Test Kit
Firstly, agarose (0.25 g) was dissolved in 10 mL of ultrapure water and heated until it was completely dissolved, obtaining a hydrogel with an agarose content 2.5% (w/v). Secondly, 20 µL of XBT-CN (10 µM) was added to the hydrogel and stirred vigorously for 1 h. Then 200 µL above mixture was poured into the centrifuge tube lid and cooled at room temperature for the sake of using the tube as a portable XBT-CN@hydrogel test kit. Subsequently, N 2 H 4 or ClO − with different concentrations was added to the centrifuge tube. After that, the centrifuge tube was turned upside down for 15 min at room temperature to allow N 2 H 4 or ClO − diffuse into the hydrogel to react with XBT-CN completely. To observe the color change of the hydrogel, the centrifuge tube was turned upside down again, and the snap cap was opened. Fluorescence images of the hydrogel in the lid under a 365 UV light were recorded by smartphone and analyzed by a color-analysis app. Finally, the N 2 H 4 or ClO − concentration can be calculated according to the proportional relationship between them and RGB values.

Detection of N 2 H 4 and ClO − in Real Samples by Hydrogel Test Kit
For the purpose of verifying the practicability of the XBT-CN@hydrogel sensor combined with a smartphone, we selected several practical samples, such as water, soil, and food. Water samples were collected from the Hebei Technology University Campus (tap water, lake water), river water was collected from the Minxin River in Shijiazhuang, and industrial wastewater was collected from a pharmaceutical enterprise in Shijiazhuang. Both soil samples from cropland, wetland, and sandland were collected in Shijiazhuang. Food samples (rice, flour, beer, and cabbage) were purchased from the Beiguo supermarket in Shijiazhuang. All the real samples were pre-treated before the measurements. Water samples were standing placed for 12 h and then filtered. Each kind of soil sample (1.0 g) was added into pure water (5 mL), stirred for 12 h, and then filtered. Food samples like solid food other than drinks were firstly digested (refer to the reported pretreatment procedures) [50][51][52]. The solid food samples (1.0 g each) were added into 0.5% NaOH solution (m/m, 10 mL), stirred overnight at room temperature, and kept refluxed for 5 h. Then the mixture was cooled to room temperature and filtered. Afterward, the HCl solution (1.0 mol/L) was dropped into the filtrate to adjust the pH to 7.0-8.0. In addition, the samples of drinks were directly adjusted to pH 7.0-8.0 by HCl solution. The procedures and conditions used for the determination of N 2 H 4 and ClO − in real samples were the same as the above Section 3.6.

Conclusions
A novel dual-response fluorescence probe XBT-CN has been successfully developed for the detection of N 2 H 4 and ClO − simultaneously. In the nucleophilic reaction of C=C bond in XBT-CN, N 2 H 4 induced the probe to produce a hydrazone structure with an obvious fluorescence change from quenched to luminous yellow. Meanwhile, It has been shown that ClO − can oxidize C=C double bond to form an aldehyde derivative with obvious fluorescence change from quenched to brilliant blue. The probe XBT-CN can be used to quantitatively determine N 2 H 4 and ClO − with a rapid response time (within 20 s) and high sensitivity. The LOD of N 2 H 4 and ClO − is 27 nM and 34 nM, respectively. It has good selectivity in a complex physiological environment. It is worth noting that we have fabricated a portable hydrogel test kit integrated with dual-emission XBT-CN for point-of-care detection of N 2 H 4 and ClO − in environmental and food samples. Attractively, XBT-CN@hydrogel can also be employed as an encryption ink for information security applications. In addition, the results of living cell imaging showed that XBT-CN had low cytotoxicity and excellent biocompatibility and could recognize and visualize N 2 H 4 and ClO − in living cells. Overall, the neoteric dual-response fluorescence probe XBT-CN successfully realizes the simultaneous and point-of-care detection of N 2 H 4 and ClO − in various fields, such as environment, food, information security, and biological imaging.